Transition state binding assay

ABSTRACT

A method to determine whether a test compound modulates the activity of an enzyme that has a metallated active site, comprising:  
     a) providing said test compound coupled to a solid support;  
     b) treating said solid support with the enzyme in metallated form;  
     c) determining as a positive result of b) that the enzyme binds to said solid support and as a negative result of b) that said enzyme does not bind to said solid support;  
     d) treating said solid support with said enzyme in a nonmetallated form;  
     e) determining as a positive result of d) that the enzyme binds to said solid support and as a negative result of d) that said enzyme does not bind to said solid support;  
     f) whereby a positive result in c) and a negative result in e) identifies said test compound as a modulator of said activity is disclosed.

RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/087,456 filed Jun. 1, 1998 and U.S. ProvisionalApplication No. 60/087,124 filed May 11, 1998, which are herebyincorporated by reference in its entirety, as if fully set forth.

TECHNICAL FIELD

[0002] The present invention is directed to an assay system forassessing whether a compound modulates the activity of an enzyme with ametallated active site. More precisely, the invention concerns a methodwhereby the compound is provided attached to a solid support and bindingof an enzyme is assessed with and without the presence of metal.

BACKGROUND ART

[0003] The use of solid supported assays for binding is well known inthe art. Various labeling methods are available whereby a compound boundto a solid support can be shown to be complexed therewith. Labelsinclude fluorescent labels, dye labels, enzyme labels, and radioisotopelabels. The labeling can be either direct—i.e., the compound tested forbinding to a ligand attached to a solid support may itself be labeled,or a secondary labeling event can be employed—e.g., a bound antibody maybe detected by a labeled antibody from another species. Such means ofdetection are well understood.

[0004] The present invention takes advantage of these mechanisms todetermine whether a compound will modulate the activity of an enzymewherein the enzyme has a metallated active site. This is accomplished bytesting the binding of the enzyme to immobilized compounds both with andwithout the presence of metal.

DISCLOSURE OF THE INVENTION

[0005] In one aspect, the invention is directed to a method to determinewhether a test compound modulates the activity of an enzyme that has ametallated active site, which method comprises:

[0006] a) providing said test compound coupled to a solid support;

[0007] b) treating said solid support with the enzyme in metallatedform;

[0008] c) determining as a positive result of b) that the enzyme bindsto said solid support and as a negative result of b) that said enzymedoes not bind to said solid support;

[0009] d) treating said solid support with said enzyme in anonmetallated form;

[0010] e) determining as a positive result of d) that the enzyme bindsto said solid support and as a negative result of d) that said enzymedoes not bind to said solid support;

[0011] f) whereby a positive result in c) and a negative result in e)identifies said test compound as a modulator of said activity.

[0012] In particularly preferred embodiments, the method is used todetermine whether a metalloprotease is modulated by a test compound andto ascertain a member of a library of compounds which will modulate theactivity of such an enzyme.

BRIEF DESCRIPTION OF THE DRAWING

[0013]FIG. 1 is a photocopy of 100×photomicrographs of beads coated withtest compounds under various conditions.

MODES OF CARRYING OUT THE INVENTION

[0014] In the assay method of the invention, the compound to be testedis first coupled to solid support. Any solid support can be used,including appropriately derivatized surfaces which may have anygeometric configuration, e.g., plates and blocks. However, in order tomaximize the efficiency of the assay, it is desirable to derivatize thecompounds to be tested on beads. A multiplicity of beads which contain amultiplicity of compounds permit more than one compound to be tested atonce.

[0015] Typical beads or resins include polyvinyl, polyvinyl chloride,polystyrene, silica, carbohydrates such as sepharose or agarose and thelike. A means for derivatizing beads of this nature are well known inthe art.

[0016] Once the compound has been derivatized to the solid support, itis treated with a metallated enzyme, whereby the compound's ability tomodulate the activity of the enzyme is to be tested. The enzyme is ametallated enzyme containing metal in the active site. This firstcontact is thus in the presence of metal. The binding of the enzyme tothe supported compound is then detected using any suitable means. Forexample, the enzyme may be biotinylated and then treated with anavidin-bound dye, or using an avidin-mediated binding. Alternatively,the enzyme may be reacted with a suitable antibody which itself bears alabel, including a fluorescent, dye, radioactive or enzyme label. Anysuitable labeling means can be used.

[0017] The solid supports where binding has been demonstrated are thentreated to remove metal ion, for example, by treating with a chelatingagent such as EDTA. Those supports where the color is retained even inthe absence of metal, contain compounds which have bound the enzyme atother than the metal-containing active site. Those solid supports whereremoval of the metal releases the enzyme contain compound which bind tothe metallated active site.

[0018] Thus, compounds that will modulate the activity of the enzymetested bind to the enzyme in the presence of metal, but not in itsabsence. Compounds that bind at irrelevant locations show similarcharacteristics both in the presence and absence of metal.

[0019] The method of the invention is especially useful in sortingcompounds in combinatorial libraries for their modulation activities.Under these circumstances, in a convenient form of the assay, thevarious members of the library are bound to individual beads. When theenzyme is contacted with the library in the presence of metal ion, or inits metallated form those beads which show label may be removed andretested in the presence of an agent that removes the metal. Those beadsthat become unlabeled in the absence of metal contain compounds thatmodulate the activity of the metal-dependent enzyme.

[0020] Although it is convenient to conduct the assay as describedabove, it is not necessary to conduct the assay sequentially—i.e., thelibrary may be divided into two portions and one portion tested forbinding to enzyme in the presence of metal ion and the other tested inthe absence of metal ion. Under such a protocol, however, an orderlyarray of the solid supports is required so that the results can becorrelated for individual solid supports. For example, the beads may beplaced in a grid so that the corresponding compounds in each case occupythe same portion of the grid or at least have a known relationship.

[0021] A large number of enzymes that contain metals in the active siteare known. Particularly important are metal-containing proteases and ina preferred embodiment, modulators of these proteases can be identifiedusing the method of the invention.

[0022] The following example is intended to illustrate but not to limitthe invention.

[0023] A multiplicity of resin beads to which is coupled the peptideCbz-Ala-X-Ala^(P)-Y-Z-Ala-Resin were prepared. In the compounds of thelibrary, X and Z represent any of 16 different amino acids and Yrepresents any of five α-hydroxy acids; Ala^(P) represents1-amino-ethylphosphonic acid, the phosphonic acid analog of alanine.Thus, the number of compounds possible in the library is 1280hexapeptides. The library was synthesized using the split-mix techniqueson 90 μm TentaGel resin. Each resin bead displays only one peptide.

[0024] The library of beads was then contacted with Botulinum A lightchain which is provided a dye label using a biotin/avidin/biotincoupling. Biotin derivatized Botulinum A light chain was prepared byalkylation of the three free cysteine residues with biotin-BMCC.Biotin-BMCC is of the formula:

[0025] The biotinylated enzyme is then labeled with avidin, furtherbound to a dye-biotin conjugate.

[0026] In the alternative, avidin itself is coupled to dyes, includinggallocyanine (blue), ethyl red (red) or carminic acid (red)5-(biotinamido)pentylamine via the standardcarbodiimide/N-hydroxysuccinimide method.

[0027] The beads were tested for nonspecific binding as follows:

[0028] Biotinylated synthesis resin was treated with avidin followed byincubation with the biotin-dye conjugates described above. Acetylatedsynthesis resin was used as a negative control. The ethyl red conjugatedbiotin showed substantial nonspecific binding to the resin—i.e., itbound to the acetylated resin as well as biotinylated resin. Of the tworemaining dye conjugates, the carminic acid conjugate gave the mostacceptable background. It bound to the biotin/avidin-labeled beads butnot to the acetylated beads.

[0029] The library of peptides coupled to beads described above wasscreened in three 40-mg batches corresponding to 100,000 beads in avolume of 120 μl.

[0030] The beads were first incubated with biotinylated Botulinum Alight-chain, then washed, then incubated with streptavidin, then washed,then treated with dye-labeled (carminic acid) biotin.

[0031] The results are showed in panel a) of FIG. 1. Most beads werecolorless but several stained a deep red. The remainder ranged fromlight pink to rose. About 150 of the colored beads were selected and setaside as a pool (shown in panel b) of FIG. 1). A subset of 20 of themost deeply colored beads from the first pool was selected and set asideas a second pool. Both pools were washed extensively with 100 mMglycine/1 mM EDTA, pH 2.5 until no color remained. The washed beads areshown in panel c) of FIG. 1.

[0032] The two pools of beads were then rescreened using Botulinum Alight-chain protein which is biotinylated but contains no zinc ion. Inthe first pool, most beads remained colorless with about 30% developinga small range of light pink coloration, as shown in panel d) of FIG. 1.In the second pool, the deep red beads quickly recovered their deep redcolor showing nonspecific binding ((panel e) of FIG. 1).

[0033] Beads that were strongly positive in the zinc containingBotulinum A light-chain binding assay and negative in the apo-BotulinumA light-chain display ligands specific for the zinc binding site of thelight chain.

1. A method to determine whether a test compound modulates the activityof an enzyme that has a metallated active site, which method comprises:a) providing said test compound coupled to a solid support; b) treatingsaid solid support with the enzyme in metallated form; c) determining asa positive result of b) that the enzyme binds to said solid support andas a negative result of b) that said enzyme does not bind to said solidsupport; d) treating said solid support with said enzyme in anonmetallated form; e) determining as a positive result of d) that theenzyme binds to said solid support and as a negative result of d) thatsaid enzyme does not bind to said solid support; f) whereby a positiveresult in c) and a negative result in e) identifies said test compoundas a modulator of said activity.
 2. The method of claim 1 wherein saidenzyme is labeled and said binding is determined by assessing thepresence, absence or amount of label associated with the solid support.3. The method of claim 2 wherein said label comprises a visible dye, afluorescent dye, an enzyme label, or a radiolabel.
 4. The method ofclaim 3 wherein said label comprises a visible dye.
 5. The method ofclaim 4 wherein said visible dye is gallocyanine, ethyl red, or carminicacid.
 6. The method of claim 2 wherein said label is bound to saidenzyme covalently through a linker moiety.
 7. The method of claim 1wherein said test compound is a member of a library of test compounds.8. The method of claim 1 wherein said solid support comprises beads. 9.The method of claim 1 wherein said enzyme is a metalloprotease.
 10. Amethod to determine whether a test compound modulates the activity of ametalloprotease which method comprises: a) providing said test compoundcoupled to a support which comprises beads; b) providing said enzymecoupled to a visible label; c) incubating said beads with said proteasewherein said protease is in metallated form; d) determining as apositive result of c) that beads retain the color of the visible dye andas a negative result of c) that said beads do not retain the color ofthe visible dye; e) incubating said beads with the labeled enzymewherein said enzyme is in nonmetallated form; f) determining as apositive result of e) that beads retain the color of the visible dye andas a negative result of e) that said beads do not retain the color ofthe visible dye; g) whereby a positive result in d) and a negativeresult in f) identify said test compound as a modulator of saidactivity.
 11. A method to detect, in a library of compound members, amember that is a modulator of the activity of an enzyme that has ametallated active site, which method comprises: a) providing saidlibrary as a multiplicity of beads, each bead coupled to molecules of anindividual member compound; b) treating at least some members of saidlibrary with said enzyme in metallated form; c) determining as apositive result of b) that the enzyme binds to beads coupled to a memberof the library and as a negative result of b) that said enzyme does notbind to beads coupled to said member of said library; d) treating beadscontaining at least some members of the library with the enzyme in anonmetallated form; e) determining as a positive result of d) that theenzyme binds to beads coupled to a member of the library and as anegative result of d) that said enzyme does not bind to beads coupled tosaid member of said library; f) whereby a positive result in c) and anegative result in e) for a given member of the library, identifies thatmember as a modulator of said activity.
 12. The method of claim 11wherein the members of the library subjected to step e) consistessentially of members of the library which give a positive result inc).
 13. The method of claim 12 wherein steps b) and c) are performedprior to steps d) and e) so as to provide a subset of members which givepositive results in step c) for testing in step d).
 14. A kit for theconduct of the method of claim 10 which kit comprises, in appropriatecontainers, a) a multiplicity of beads wherein each bead is coupled tomolecules of a single member of a library of test compounds; and b)either a metallated enzyme of interest coupled to a dye or an activatedlabel.